Misfolded and potentially harmful proteins that accumulate at the endoplasmic reticulum (ER) are destroyed via one of several branches of ER-associated degradation (ERAD). The recently identified ERAD-T (translocon) pathway in Saccharomyces cerevisiae catalyzes the destruction of proteins that persistently or aberrantly engage the ER translocon (the channel responsible for moving proteins across the ER membrane). ERAD-T, which is mediated by the Hrd1 ubiquitin ligase, is specifically impaired under conditions of ER stress (an elevated burden of unfolded proteins in the ER) via an uncharacterized ER stress response. Recent evidence strongly suggests that the protein component of low-density lipoproteins (LDL, i.e. bad cholesterol) is degraded via ERAD-T when its translocation into the ER has stalled. In addition, many diseases are associated with protracted ER stress (including certain cancers, neurodegenerative conditions, and immune disorders). Pharmacologically manipulating the protein folding and quality control capacity of the ER may be a common therapeutic strategy for such conditions. Consistent with the mission of the National Institute of General Medical Sciences, the long-term objective of the proposed work is an improved understanding of ERAD-T, a cellular quality control mechanism about which virtually nothing is known. Insights about this pathway are highly likely to inform an understanding of LDL physiology and the development of improved therapeutic strategies for cholesterol- and ER-stress-related pathologies. The proposed studies will address two hypotheses: (1) interaction of the Hrd1 ubiquitin ligase with the translocon is required for degradation of ERAD- T substrates, and (2) components of a novel ER stress-sensing mechanism participate in ERAD-T and become limiting for ERAD-T under ER stress conditions. The specific aims of this project are to (1) identify mutations that specifically impair ERAD-T and (2) investigate the relationship between ER stress and ERAD-T. Novel, genetic assays that indicate whether ERAD-T is functional on the basis of yeast cell growth will be conducted and biochemical analyses will be performed to identify and characterize the genetic requirements for ERAD-T. This work will expose undergraduates and master's students to meaningful, biomedically relevant research. Students will participate in every aspect of this project, includin experimental design and execution, interpretation of results, and data presentation. Students will gain valuable firsthand experience in biomedical research and have the chance to make new discoveries about an area of basic biology with important medical implications.